The Process of Protein Synthesis
Protein synthesis is one of the most central life processes. A protein is formed by the linkage of multiple amino acids via peptide bonds, according to a sequence defined by the template messenger RNA (mRNA). Protein synthesis occurs in the ribosomes, the protein manufacturing plants of every organism and nearly every cell type.
Ribosomes are ribonucleoprotein particles consisting of a small and large subunit. In bacteria these subunits have sedimentation coefficients of 30 and 50, and thus are referred to as “30S” and “50S” respectively; in eukaryotes the sedimentation coefficients are 40 and 60. The translation system makes use of a large number of components, including inter alia the ribosome, initiation, elongation, termination and recycling factors, transfer RNA, amino acids, aminoacyl synthetases, magnesium, and the product polypeptides.
Aminoacylated tRNAs read codons in ribosome-bound messenger RNAs and transfer their attached amino acids to growing peptide chains. The tRNA molecule has 73-93 nucleosides arranged in a cloverleaf-like structure, and includes the elements of an acceptor stem, a D-loop, an anticodon loop, a variable loop and a TψC loop. Aminoacylation, or charging, of tRNA results in linking the carboxyl terminal of an amino acid to the 2′-(or 3′-) hydroxyl group of a terminal adenosine base via an ester linkage. Aminoacylation occurs in two steps, amino acid activation (i.e. adenylation of the amino acid to produce aminoacyl-AMP), tRNA aminoacylation (i.e. attachment of an amino acid to the tRNA).
In most species, multiple tRNA molecules exist which become aminoacylated with the same amino acid but have different anticodon sequences. Such families of tRNAs are termed isoacceptors. There are 21 isoacceptor families, corresponding to 20 for the standard amino acids and one for seleno-cysteine. A particular tRNA may be denoted according to its aminoacylating amino acid, also referred to as its cognate amino acid, which is indicated in superscript such as in tRNALeu. A particular tRNA may further be denoted according to its anticodon, indicated in parentheses, such as tRNALeu (CAG).
According to the genetic code, the total possible number of anticodons is 64, meaning that 61 different tRNA species (minus the stop codons) are theoretically required for translation. However, in most organisms the total number of tRNA species is generally less than this. This is due to the fact that at least some tRNAs are capable of reading (or “decoding”) different codons that encode the same amino acid. For example, E. coli has five tRNALeu isoacceptors that decode the six leucine codons, and four tRNAArg isoacceptors that decode the six arginine codons. Distinct codons that encode the same amino acid are termed “synonymous” codons. Synonymous codons which differ only at the third base may often be read by the same tRNA due to “wobble” pairing. Wobble pairing is base pairing between a codon and an anticodon that does not obey the standard Watson-Crick pairing at the wobble position.
Protein translation, also referred to as “polypeptide synthesis,” involves the stages of initiation, elongation and termination. The initiation stage begins by formation of the initiation complex, composed of the two ribosomal subunits, protein initiation factors, mRNA, and an initiator tRNA, which recognizes the initiator codon UAG of open reading frames. Elongation proceeds with repeated cycles of charged tRNAs binding to the ribosome (a step termed “recognition”), peptide bond formation, and translocation, involving elongation factors and enzymes such as peptidyl transferase, which catalyzes addition of amino acid moieties onto the growing chain. Termination factors recognize a stop signal, such as the base sequence UGA, in the mRNA, terminating polypeptide synthesis and releasing the polypeptide chain and mRNA from the ribosome. Recycling factor enables dissociation of the ribosome subunits, which are then available for a new round of protein synthesis (see for example, Kapp et al., 2004, Annu Rev Biochem. 73:657-704). In eukaryotes, ribosomes are often attached to the membranes of the endoplasmic reticulum (ER) and Golgi compartments. Additionally, ribosomes are active in organelles such as mitochondria and, in plant cells, in chloroplasts, and in other subcellular compartments. One important locus of protein synthesis activity is in dendritic spines of neurons.
Stem Cell-Based Therapies for Congenital Diseases
Stem cells and stromal cells of various types are currently in development for potential use in cell therapy for a wide variety of congenital diseases involving a deficiency of one or more proteins involved in normal growth or metabolism. Such proteins include insulin, growth factors, and erythropoietin, among many others. Stem and stromal cell populations that secrete such proteins in therapeutic qualities are urgently sought after, as described for example in Soto-Gutierrez A et al, Acta Med Okayama. 2008 April; 62(2):63-8; and in Yokoo T et al, Transplantation. 2008 Jun. 15; 85(11):1654-8. Methods for monitoring production of the target protein from stem cell candidates are critical to the development of such therapies.
In addition, stem and stromal cells have great potential in regenerative therapy for a wide variety of disorders, as described in Caplan A I., Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol. 2007 November; 213(2):341-7. In order to monitor differentiation of stem and stromal cells into the desired cell types, it is important to be able to measure production of specialization and differentiation markers and factors at the protein translation level.
Use of Cells for Antibody Production
Cultured cells are widely used for production of recombinant antibodies for various therapeutic uses. In order to optimize antibody production, it is important to be able to measure antibody production at the protein translation level.
Fast and efficient methods for measuring translation of a particular protein of interest in real time are particularly useful in the above applications relating to stem cells and antibody production.
Diseases Related to Protein Translation
Control of protein translation is implicated in a large number of diseases. For example, the family of central nervous system (CNS) disorders connected with protein synthesis disturbances in neural spines. The family includes fragile X mental retardation, autism, aging and memory degeneration disorders such as Alzheimer's disease. Neural spines and synapses contain their own protein synthesis machinery. Synaptic plasticity, underpinning the most basic neural functions of memory and learning, is dependent upon proper regulation of spinal protein synthesis. Another important family of diseases directly connected to protein synthesis includes genetic disorders associated with the presence of premature termination codons (PTC) in the coding sequence of a critical protein, preventing its translation. Such diseases include Duchenne Muscular Dystrophy and a large family of congenital diseases. A small molecule known as PTC124 (Welch E M et al, Nature 2007 May 3; 447(7140):87-91) helps the ribosome slide over the mutated codon, thereby producing the required protein, albeit at only at 1-5% of normal concentrations. These amounts are often sufficient to sustain the life of an afflicted individual. PTC suppression has also been achieved by introducing charged suppressor tRNA into a living cell, enabling readthrough suppression of the PTC-containing mRNA and accumulation of the encoded protein (Sako et al, Nucleic Acids Symp Ser, 50:239-240, 2006.
Multiple myeloma (MM) is a cancer of the plasma cells characterized by proliferation of malignant plasma cells and a subsequent overproduction of intact monoclonal immunoglobulin (IgG, IgA, IgD, or IgE) or Bence-Jones protein (free monoclonal κ and λ light chains). This incurable disease accounted for a disproportionate 2% cancer-related death rate in the United States in 2008 (Jemal A, et al. Cancer statistics, 2008. CA Cancer J Clin. 2008; 58:71-96). While current treatments including autologous transplantation (Harousseau J L, et al. Best Pract Res Clin Haematol. 2005; 18:603-618) have resulted in an improvement in overall survival (Kumar S K, et al. Blood. 2008; 111:2516-2520), there remains a need for delineating MM cell biology. It has been disclosed that overall proteasome activity of primary MM cells inversely correlates with apoptotic sensitivity to proteasome inhibition (Bianchi G et al. Blood, 26 2009, Vol. 113, No. 13, 3040-3049). The associated mechanism apparently involves triggering of stress responses which are characterized by an increase in the clearance of misfolded proteins by the 26S proteasome, an increase in the transcription and translation of chaperone proteins and foldases, and a strong decrease in global protein synthesis (Fribley A, et al. 2006. Cancer Biol Ther 5: 745-748; Bush K T, et al. J Biol Chem 272 (14) 1997 9086-9092.
Hepatic fibrosis is a common response to most chronic liver injuries, including viral hepatitis, parasitic infection, metabolic and immune diseases, congenital abnormalities, and drug and alcohol abuse. It is characterized by increased production of fibril-forming collagen and associated scar formation. Collagen is a family of proteins, wherein the constituent chains are generally about 300 amino acids in length, and are highly enriched in Gly-Pro and Pro-Gly repeating units. Currently, collagen is measured with DNA microarrays, which indicate the existence of collagen transcripts, but do not correlate well with actual synthesis of the protein.
In each of the above disease applications, it is important to rapidly and efficiently measure in real-time translation of a one or more particular proteins of interest. In some cases, it is important to study the subcellular localization of such proteins, as for example in the case of local protein synthesis in neurons.
FRET Technology
Fluorescence resonance energy transfer (FRET) is a method widely used to monitor biological interactions. FRET utilizes a donor fluorophore, having an emission spectrum that overlaps with the excitation spectrum of the acceptor fluorophore. Only when the donor fluorophore and acceptor fluorophore are in close proximity, typically about 10 nm, is a signal emitted from the acceptor fluorophore. FRET is described in Szöllosi J, Damjanovich S, Mátyus L, Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research, Cytometry 34(4):159-79, 1998.
Existing Methods of Measuring Protein Translation
Cell assays that quantify production of a single specific protein typically require cell lysis and/or permeabilization, making then unsuitable for analysis of living cells. Further required are antibodies or other specific reagents directed to the protein of interest in conjunction with various dyes and detection reagents. Further, such methods produce an estimation of the total production of proteins over a given period of time measured in minute, hours or days. Those methods which are suitable for intact cells and organelles, such as metabolic labeling with radioactive tracers, are unable to isolate a signal from a particular transcript or protein of interest, and thus do not provide real-time or dynamic measurements of translation activity.
U.S. Pat. No. 6,210,941 to Rothschild discloses methods for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system. tRNA molecules are mis-aminoacylated with non-radioactive markers that may be non-native amino acids, amino acid analogs or derivatives, or substances recognized by the protein synthesizing machinery. U.S. Patent Application Publication Nos. 2003/0219783 and 2004/0023256 of Puglisi disclose compositions and methods for solid surface translation, where translationally competent ribosome complexes are immobilized on a solid surface. According to the disclosure, ribosomes and one or more components of the ribosome complex may be labeled to permit analysis of single molecules for determination of ribosomal conformational changes and translation kinetics.
WO 2004/050825 of one of the inventors of the present invention discloses methods for monitoring the synthesis of proteins by ribosomes in cells or a cell-free translation system, involving use of donor and acceptor fluorophores to label two locations of a ribosome, or each of a ribosome and a tRNA, or each of a ribosome and an amino acid.
WO 2005/116252 of one of the inventors of the present invention discloses methods for identifying ribonucleotide sequences of mRNA molecules using immobilized ribosomes in a cell-free translation system. In the disclosed methods, a tRNA and a ribosome component are engineered to carry donor and acceptor fluorophores or are used in FRET via their natural fluorescent properties.
The prior art does not disclose or suggest a method for measuring rates of translation of a protein of interest in living cells which utilizes a pair of tRNA molecules directly labeled with two components constituting a FRET pair, wherein the pair of tRNAs corresponds to a specific pair of adjacent amino acids occurring in the protein. There is an ongoing need for such methods. Methods for measuring changes in protein synthesis rates in response to a drug candidate would be very useful for drug screening and assays for predicting therapeutic activity of candidate drugs. Also highly advantageous would be methods for measuring translation of a protein of interest that are amenable for high-throughput screening and cell sorting.